Multifunctional biosensor based on ZnO nanostructures

ABSTRACT

The present invention provides the multifunctional biological and biochemical sensor technology based on ZnO nanostructures. The ZnO nanotips serve as strong DNA or protein molecule binding sites to enhance the immobilization. Patterned ZnO nanotips are used to provide conductivity-based biosensors. Patterned ZnO nanotips are also used as the gate for field-effect transistor (FET) type sensors. Patterned ZnO nanotips are integrated with SAW or BAW based biosensors. These ZnO nanotip based devices operate in multimodal operation combining electrical, acoustic and optical sensing mechanisms. The multifunctional biosensors can be arrayed and combined into one biochip, which will enhance the sensitivity and accuracy of biological and biochemical detection due to strong immobilization and multimodal operation capability. Such biological and biochemical sensor technology are useful in detection of RNA-DNA, DNA-DNA, protein-protein, protein-DNA and protein-small molecules interaction. It can be further applied for drug discovery, and for environmental monitoring and protection.

CROSS-REFERENCE TO RELATED APPLICATION:

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/385,884, which was filed on Jun. 6, 2002.

This invention was made with Government support under Grant Nos. NSFECS-0088549 and NSF CCR-0103096, awarded by the National ScienceFoundation. Therefore, the Government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates generally to biosensor technology, and pertainsmore particularly to novel multifunctional biosensors based on zincoxide (ZnO) nanostructures for biological, biochemical, chemical andenvironmental applications.

BACKGROUND OF THE INVENTION

The nanoscale science and engineering have shown great promise for thefabrication of novel nano-biosensors with faster response and highersensitivity than that of planar sensor configurations, due to theirsmall dimensions combined with dramatically increased contact surfaceand strong binding with biological and chemical reagents which couldhave important applications in biological and biochemical research, aswell as in environmental monitoring and protection.

ZnO nanostructures have many advantages. As disclosed in U.S. patentapplication Ser. No. 10/243,269, nanotip arrays made with insulating orconductive ZnO can be fabricated in a controlled manner to produce tipswith a uniform size, distribution and orientation. The ZnO nanotips aremade using our chemical vapor deposition (CVD)-based method in a simpleprocess at relatively low temperatures as disclosed by S. Muthukumar*,H. Sheng*, J. Zhong*, Z. Zhang*, N. W. Emanaetoglu*, Y. Lu, “SelectiveMOCVD Growth of ZnO Nanotips”, IEEE Trans. Nanotech, Vol. 2, n. 1, pp.50-54 (2003), giving ZnO nanostructures a unique advantage over otherwide bandgap semiconductor nanostructures, such as gallium nitride (GaN)and silicon carbide (SiC). Furthermore, through proper doping andalloying, ZnO nanotips can be made as piezoelectric and ferroelectric,transparent and conducting, and magnetic, thus having multifunctionalapplications.

Recent advances in genetic sequencing methods are leading to anexplosion in the area of biotechnology. Many emerging areas ofbiotechnology are based upon highly-parallel methods for sequencing anddetecting DNA, RNA, and proteins. Many of these areas could benefitgreatly by leveraging the emerging nanotechnology, but applying it todevelop and utilize new analytical tools for biochemical analysis. Aneed exists to provide novel biological and biochemical sensors, whichhave higher sensing efficiency and multiple functionality, therebyhaving significant advantages in comparison to the existing sensortechnology.

SUMMARY OF THE INVENTION

It is the primary objective of this invention to address the novelmultifunctional biosensor technology based on ZnO nanotips and nanotiparrays.

Particularly, it is an objective of this invention to provideconductivity-based biosensors using semiconductive or conductive ZnOnanotips; to provide field-effect-transistor (FET)-based biosensors byusing ZnO nanotips as the gate of the FET; to provide surface acousticwave (SAW)-based biosensors by integrating ZnO nanotips into SAW devicesto form highly sensitive and multichannel biosensors; and to providebulk acoustic wave (BAW)-based biosensors by integrating ZnO nanotipsinto BAW devices to form highly sensitive and multichannel biosensors.

As ZnO nanotips can be made semiconducting, transparent and conducting,or piezoelectric, their unique electrical, optical and acousticproperties can serve as the basis for multifunctional sensors. A sensorchip comprising of arrays and combinations of various types of ZnOnanotip-based biosensors also allow for multimodal operation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows a schematic of a vertical cross-section view of thedevice structure for the conductivity-based ZnO nanotip biosensor.

FIG. 1 b shows a schematic of top view of the conductivity-based ZnOnanotip biosensor structure.

FIG. 2 shows a schematic of a vertical cross-section of a ZnO nanotipgate metal-insulator-semiconductor field effect transistor (MISFET).

FIG. 3 a shows a schematic of a vertical cross-section view of a ZnOnanotip SAW sensor.

FIG. 3 b shows a schematic of a top view of ZnO nanotip SAW sensor.

FIG. 4 a shows an example of transmission spectra as a function offrequency for test ZnO nanotip SAW sensor devices.

FIG. 4 b shows a plot of a phase shift as a function of frequencybetween the reference and test ZnO nanotip SAW sensor devices.

FIG. 5 shows a schematic of a vertical cross-section of a ZnO nanotipBAW sensor.

DETAILED DESCRIPTION OF THE TECHNOLOGY

A biosensor is a device which is capable of providing analysis ofvarious analytes or biomolecules using biological recognition elementswhich are combined with a signal transducer. Generally, the sensor willproduce a signal that is quantitatively related to the concentration ofthe analytes.

The biological recognition elements serve to recognize the analytes.These elements include enzymes, microorganisms, tissues, antibodies,receptors, nucleic acids, organelles or whole cells.

Transducers are physical components of the biosensor that respond to theproducts of the biosensing process and outputs the response in a formthat can be amplified, stored or displayed. Biosensing occurs only whenthe analyte is recognized specifically by the biological element.Biological recognition in vivo at a single cell level is characterizedby high sensitivity, fast response, specificity and reversibility.

A “sensor surface” refers to the location upon which a binding partneris immobilized for the purpose of measuring changes in physicalproperties, such as optical refractive index, electrical conductivity,mass loading, etc. They include, but are not limited to, semiconductor,metal and dielectric surfaces.

ZnO is a wide bandgap semiconductor having a direct bandgap of 3.32 eVat room temperature and can be made semiconducting, piezoelectric,ferroelectric, ferromagnetic, and transparent and conducting throughproper doping. ZnO has an exciton binding energy of 60 meV. It is foundto be significantly more radiation hard than silicon (Si), galliumarsenide (GaAs), and GaN.

ZnO is a polar semiconductor with the (0002) planes being Zn-terminatedand the (000{overscore (2)}) planes being O-terminated. These twocrystallographic planes have opposite polarity and hence have differentsurface relaxations energies. This leads to a higher growth rate alongthe c-axis. The ZnO film grown on many semiconducting, insulating ormetallic substrates have a preferred c-axis orientation normal to thesurface. Therefore, ZnO growth results in a pillar like structure calledZnO nanotips on these semiconducting, insulating and metallicsubstrates, while ZnO grown on R-plane sapphire substrates results in asmooth epitaxial film. The ZnO nanotips can be grown at relatively lowtemperatures, giving ZnO a unique advantage over other wide bandgapsemiconductor nanostructures, such as GaN and SiC.

ZnO is an important multifunctional material, which has wideapplications in telecommunications, chemical and biochemical sensors andoptical devices. In this application, ZnO nanotips are used as thesensor surface to enhance the immobilization in detection of DNA,protein, and harmful biological agents in the field of biological andbiochemical sensors. The use of ZnO nanotip arrays also greatlyincreases the effective sensing area of the biosensor devices as will bedescribed in greater detail below.

ZnO nanotips can be grown on various substrates. They can also beselectively grown on patterned layers of materials through substrateengineering. Both cases have been disclosed in U.S. patent applicationSer. No. 10/243,269.

Referring to FIGS. 1 a and 1 b, a schematic of vertical cross-sectionview and a top view respectively of a conductivity-based ZnO nanotipbiosensor 10 are shown. The biosensor consists of a substrate 16, aconductive thin film 14, a ZnO nanotip array 12 on the conductive thinfilm 14, and metal electrode pads 17. A reaction between the immobilizedspecies on ZnO nanotips 12 with the target will result in a change inthe total change accumulated on nanotips. This change will cause atransient current across the biosensor device, which will be used as thesensor output, as will be described below.

The substrate 16 can be a semiconductor substrate, such as Si or GaAs,in which case the biosensor can be integrated with electronic integratedcircuits (ICs). In a second embodiment of the invention, the substrate16 can be a transparent insulating substrate, such as glass or sapphire,in which case both electrical and optical sensing mechanisms can be usedto realize a multifunctional sensor. In a third embodiment of theinvention, the substrate 16 can be a piezoelectric substrate, such asquartz or lithium niobate (LiNbO₃), in which case the conductivity-basedsensor can be integrated with the SAW-based sensor to realize anothertype of multifunctional sensor as described later in this application.

The conductive thin film 14 has certain conductivity, and it can be asemiconductor, such as Si with properly designed doping level, ametallic thin film, such as gold (Au), a transparent conductive oxide,such as indium tin oxide (ITO), or even the multilayer thin film. Thethin film and the metal bond pads are deposited on the substrate 16,then patterned using the standard microelectronic processing techniques.

The ZnO nanotips 12 can be deposited on the substrate 16 and thin film14 using the technology, but not limited to metal-organic chemical vapordeposition (MOCVD), then patterned by the standard photolithography andetching process.

These ZnO nanotips serve as DNA or protein molecule binding sites. Inother words, the ZnO nanotips 12 are preferably bonded with protein orDNA molecules to make conductivity-based biosensors, as will bedescribed in detail below. Specifically, the conductive thin film 14surface, with ZnO nanotips 12 grown on the top, will be designed andfabricated as conductivity-based biosensors. Preferably a probe isattached to said tip to seek the targeted molecule due to bioreaction.The probe may preferably be attached on a binding site or a targetmolecule preferably has a probe. Any useful probes preferably such aschemiluscence, fluorescence, etc. The dimensions of the conductivepattern, the aspect ratio and doping level of the ZnO nanotips, areoptimized to enhance the sensitivity. Due to depletion or accumulationof carriers in the nanotips as a result of bioreactions, the conductanceof the patterned tip arrays will change significantly. The depletion(accumulation) of the nanotips will result in a transient current acrossthe line. The amplitude of this current will be a function of the amountof target material detected, and the duration to the reaction time. Thesimilar effect was recently demonstrated using Boron doped siliconnanowire biosensors for detection of protein-protein interactions by Y.Cui, Q. Wei, H. Park, and C. M. Lieber, “Nanowire nanosensors for highlysensitive and selective detection of biological and chemical species”,Science 293, 1289 (2001).

If a transparent substrate, either insulating or piezoelectric, is used,the conductivity based ZnO nanotip biosensor 10 can be operated inoptical mode simultaneously with the conductivity mode. ZnO has anoptical cut-off wavelength of approximately 373 nm at room temperature.This optical cut-off wavelength can be extended by using its ternarycompound, magnesium zinc oxide (Mg_(x)Zn_(1-x)O). As MgZn_(1-x)O istransparent down to 240 nm (for x=0.6), the changes in the opticalabsorption characteristics before and after the bioreactions can also bedetected and analyzed. During operation, the device is illuminated withultra-violet (UV) light from one side (top or bottom), and the light isdetected on the other side. Changes in the UV absorption spectra areunique to each chemical, allowing identification of the reactantspecies. Further more, if the tip array is coated with a thin layer ofAu (<100A), it can also be functional for fluorescence biosensing asshown by V. H. Perez-Luna, S. Yang, E. M. Rabinovich, T. Buranda, L. A.Sklar, P. D. Hampton, and G. P. Lopez, “Fluorescence biosensing strategybased on energy transfer between fluorescently labeled receptors and ametallic surface”, Biosens Bioelectron. 17, 71 (2002).

In another embodiment of the present invention, there is disclosed asecond type of device, which is a ZnO nanotip-gatefield-effect-transistor (FET). FETs have been used for chemical sensors.In a FET, a voltage bias applied to the gate of a FET will modulate thecurrent flowing between its source and drain. There are two major typesFETs which can be used with biosensors. The first is ametal-insulator-semiconductor FET (MISFET), composed of a metal gatedeposited on a gate insulator layer, which is deposited on thesemiconductor. The second is a metal-semiconductor FET (MESFET),composed of a metal gate directly deposited on the semiconductor. If thegate insulator is specifically an oxide, the MISFET device is known as ametal-oxide-semiconductor FET (MOSFET).

An FET type of biosensor can be realized by depositing ZnO nanotips onthe gate region of the FET. Such an FET can be a current existing SiMOSFET, GaAS MESFET, etc. The surface charge changes occurring with thetarget on the ZnO nanotips will result in a potential difference betweenthe gate and the substrate, and modulate the current flowing between thesource and the drain. Unlike the resistor-type conductivity-based sensordescribed above, the FET type sensor can be used for both transient andsteady-state current measurements, making it a more flexible device.

More specifically, a novel transparent FET sensor is composed of a ZnOnanotip gate and a ZnO FET. Referring to FIG. 2, there is shown aschematic of a vertical cross-section view of nanotip gate ZnO MISFETbiosensor 20. It is composed of a R-plane sapphire (R—Al₂O₃) substrate22, a semiconductor ZnO thin epitaxial layer as a channel 24, doped ZnOsource and drain regions 25, a gate insulator 26, metal electrodes 27 tothe source and drain regions 25, the ZnO nanotips 12 deposited on thegate, and an encapsulation layer 28 to protect the device except thenanotip gate area.

In this device, n⁺-ZnO 25 regions serve as the source and the drain.When Al is used for the metal contacts 27, it will heavily dope the ZnOthin film 24 under it, resulting in good non-alloyed ohmic contact asdeveloped in H. Sheng, N. W. Emanetoglu, S. Muthukumar, and Y. Lu,“Non-alloyed Ohmic Contacts to Mg_(x)Zn_(1-x)O”, J. ElectronicMaterials, 31 (2002). This process will be used to simultaneously dopethe source and drain regions and form their ohmic contacts in the ZnOMISFET structure. A thin insulation layer 26 will be deposited, andpatterned on top of the n-ZnO 24 thin film. Candidate insulators 26include, but are not limited to, silicon dioxide (SiO₂) and magnesiumzinc oxide (Mg_(x)Zn_(1-x)O) with more than 50% Mg mole percentagecomposition. The device will be protected with the encapsulating layer28 from the chemical environments it operates in. The ZnO nanotips 12will be grown on the insulator layer 26, patterned and etched to serveas the nanotip-gate.

The operation of the ZnO nanotip-gate transparent MISFET 20 is simple.When a biological reaction occurs with the target at the ZnO nanotips12, the negative surface charge will change, inducing with a potentialdifference between the gate and the ZnO film. This potential differencewill change the conductivity in the n-type ZnO channel 24 under the gateinsulator 26, resulting in a change in the current between the sourceand the drain regions 25.

As in the conductivity-type ZnO nanotip biosensors 10 described above,the ZnO nanotip-gate transparent MISFET biosensor 20 can be operated inoptical mode simultaneously with electrical mode. ZnO nanotips, R—Al₂O₃,SiO₂ and Mg_(x)Zn_(1-x)O (0.5<x<1) are all transparent to visible light,therefore allow the sensor to be operated in optical mode.

In another embodiment of the present invention, there is disclosed athird type of device which integrates piezoelectric ZnO nanotips withSAW biosensors. Referring to FIGS. 3 a and 3 b, there is shown aschematic of a vertical cross-section view and a schematic of a top viewrespectively of ZnO nanotip SAW biosensor 30. The ZnO nanotip SAWbiosensor is composed of a piezoelectric substrate 32, an insulatingamorphous layer 34, a metal input interdigital transducer (IDT) 36, ametal output IDT 38, and the ZnO nanotips 12.

The piezoelectric substrate 32 can be, but is not limited to, quartz,LiNbO₃, lithium tantalate (LiTaO₃), etc. An insulating amorphous layer34 is deposited on the piezoelectric substrate and patterned using thestandard microelectronic processing techniques. This insulatingamorphous layer can be, but is not limited to, SiO₂ or Si₃N₄.

The ZnO nanotips 12 are deposited on the surface of the insulating layer34 using MOCVD, or other deposition technology, then patterned andetched to define the nanotip coverage area. The metal IDTs 36 and 38 arethen deposited and patterned using standard microelectronic processingtechniques. The metal of choice is Al, but other metals can also beused.

The ZnO nanotip SAW sensor device 30 operates similarly to a planar SAWbiosensor. A dualchannel biosensor consisting of two identical devices,one without target coating serving as the reference and the other withtarget coating serving as the sensor, are used together. As the targetbinds with the ZnO nanotips 12 on the sensor device, mass loading of thesensor will result in a decrease of the phase velocity under the ZnOnanotips. This will results in a phase difference between the outputsignals of the reference and the sensor devices. The use of ZnO nanotipsdramatically enhances the immobilization of DNA, protein and other smallbiomolecules, therefore the sensitivity of the biosensors. Ourpreliminary experimental results demonstrate that the immobilizationrate of ZnO nanotips is over thirty times higher than that of smoothsurface. It is well known that the rough surface will increase theviscosity of the sensing media on the acoustic path and deteriorate thedevice performance. Therefore, the ZnO nanosize tip-type structures 12will have higher electromechanical coupling coefficient to compensatethe increased propagation loss as disclosed by K. K. Zadeh, A. Trinchi,W. Wtodarski, and A. Holland, “A novel love-mode device based on aZnO/ST-cut quartz crystal structure for sensing applications”, Sensorsand Actuators A 3334, 1 (2002).

In the refinement of the invention, the SAW based ZnO nanotip biosensor30 can be operated in optical and SAW modes simultaneously, if atransparent piezoelectric substrate, such as quartz or LiNbO₃, is used.As in the conductive-type nanotip biosensors 10, the sensor 30 isilluminated with UV light on one side (either top or bottom) and thetransmitted UV light is detected at the other side. The UV absorptionspectrum can be used to identify the reactant species.

In another refinement of the invention, the SAW based ZnO nanotipbiosensor 30 can be operated in electrical and SAW modes simultaneously,if the insulating layer structure is replaced with the resistor-typeconductive ZnO nanotip biosensor structure.

In a further refinement of the invention, the ZnO nanotips 12 can becombined with the monolithically integrated tunable SAW (MITSAW) sensorsdisclosed previously (U.S. Pat. No. 6,559,736 and U.S. patentapplication Ser. No. 09/905,205), to enhance their performance.

In preliminary work, SAW delay lines are fabricated on 128° Y-cut LiNbO₃with a ZnO nanotip/SiO₂ layer structure deposited on the propagationpath. The IDT structure of this prototype device consists of 50 pairs ofelectrodes, 963 μm long, 3 μm wide and 3 μm apart from each electrodefor both IDTs. The phase velocity (v) of the SAW on the 128° Y-cutLiNbO₃ is 3668 m/s, and the wavelength (λ) of the test pattern is 12 μm.From the equation f_(c)=v/λ, the expected center frequency is 305 MHz.The bandwidth is BW_(3db)=(0.9/Np)*f_(c)=0.9*305/50=5.49 MHz. On thepropagation path of the prototype devices, the sensor region has 600 nmZnO nanotip/100 nm SiO₂ is 1116 μm long and 594 μm wide. Furthermore,the dual channel (reference and sensor channels) device is tested usingan Agilent 8573D Network Analyzer. The reference channel has no proteinbonding and the sensor channel is bonded with 100 ng protein on the ZnOnanotip over an area of 6.629×10⁻³ cm².

The frequency responses of the reference sensor device and the actualtest sensor device are shown in a graph shown in FIG. 4 a. The X-axis isthe frequency and the Y-axis is the S₂₁ transmission spectra ofreference and sensor device. As shown in FIG. 4 a, the sensor device hasa shift to lower frequency compared with the reference device. Anadditional insertion loss of 6.14 dB is observed for the protein bondedsample. However, the insertion loss shift depends on a number offactors, and by itself is not a good sensing mechanism. Instead, thephase shift of the signal is preferred for accurate and repeatablemeasurements.

FIG. 4 b shows a graph displaying a phase difference between thereference and sensor prototype devices. The X-axis is the frequency andY-axis is the phase difference between the reference and sensor device.As shown in FIG. 4 b, the sensor device has a 47.680 phase shift at thecenter frequency 305 MHz compared with the reference device. The phaseshift increases with increasing frequency, due to the different velocitydispersion characteristics of the SAW propagating in the reference andsensor channels. The phase velocity decreases with increasing frequency,as sensitivity increases with frequency. This phase velocity decreaseresults with a larger phase difference between the sensor and referencedevices with increasing frequency. Nominally, the phase shift should bea monotonically increasing function. However, due to such factors aselectromagnetic feedthrough and triple transit interference (TTI), thephase response has ripples. The impact of these secondary effects onsensor performance can preferably be minimized. The proof-of-conceptdevice uses an unoptimized SAW delay line structure. Narrower bandwidthsand larger phase shifts can be achieved by optimizing the deviceparameters and proper choice of substrate.

In another embodiment of the present invention, there is disclosed afourth type of device which integrates piezoelectric ZnO nanotips withBAW biosensors. Referring to FIG. 5, there is shown a schematic of avertical cross-section view of a ZnO nanotip BAW biosensor 50. The ZnOnanotip BAW biosensor 50 is composed of a piezoelectric material 52, ametal top electrode 54, a metal bottom electrode 56 and ZnO nanotips 12.

The piezoelectric material 52 can be, but is not limited to, quartz,LiNbO₃, LiTaO₃, etc. The metal top 54 and bottom 56 electrodes aredeposited and patterned using the standard microelectronic processingtechniques.

The ZnO nanotips 12 are deposited on the top metal electrode surfaceusing MOCVD, or other deposition technology, and then patterned andetched to define the nanotip coverage area of the BAW sensor.

In a further embodiment of the ZnO nanotip BAW sensor 50, the centerarea of the top surface of the piezoelectric substrate is notmetallized. The ZnO nanotips 12 are deposited on the bare piezoelectricsubstrate surface 52, so that the top metal electrode 54 surrounds, butdoes not contact the ZnO nanotips 12.

The ZnO nanotip BAW sensor 50 operates similarly to a BAW resonatordevice. The BAW resonator will resonate at a specific frequencydetermined by the piezoelectric substrate material properties andthickness. When bonding of the target occurs on the ZnO nanotips 12,mass-loading results with a shift in the resonance frequency of theresonator, directly proportional to the amount of target material bondedto the ZnO nanotips 12.

In a further embodiment of the ZnO nanotip BAW sensor 50, the crystalresonator of FIG. 5 can be replaced with a thin film resonatorstructure, including, but not limited to, air gap resonators, solidlymounted resonators and membrane (Film Bulk Acoustic Resonator or FBAR)resonators. The thin film resonator structure includes, but not limitedto, an air-gap structure on top surface of the substrate, a membranestructure on the substrate and an acoustic mirror on top surface of thesubstrate.

In a further embodiment of the present invention, there is disclosed abiochip consisting of ZnO nanotip array as biosensors to simultaneouslydetect a number of different biological information. For certainclinical and scientific applications it is desirable to use multiplebiosensors on a chip for simultaneous detection of several biomoleculartargets. For this purpose a biosensor chip having multiple detectionunits for different targets. As described above, both types of biosensordevices with 1D or 2D arrays on a chip can be fabricated through regularmicroelectronics fabrication processes.

The current invention presents a new biochemical sensor technology withhigh 20 sensitivity and multimodal operation capability. The sharp ZnOnanotips 12 on four type devices, as discussed above, provide thefavorable binding sites to enhance the immobilization, and increase theeffective sensing area, therefore, improve the sensing and detectionefficiency.

The changes in electrical conductivity, or/and optical absorption,or/and fluorescence in conductive and semiconductive ZnO nanotips willbe used to sense the targeted biochemical reactions. The ZnO nanotip SAWor BAW sensors possess both the advantages of SAW or BAW, andnanostructured biosensors.

The ZnO biosensors described above are used to detect RNA-DNA, DNA-DNA,protein-protein, protein-DNA, and protein-small molecule interactionstaking advantage of the enhancement of immobilization of DNA, proteinmolecules on the ZnO nanotips. The optimum immobilization conditions canbe used for the biosensors to further enhance their sensitivities andspecify the target molecules.

For DNA immobilization, a solution of avidin is applied to the cleansurface of ZnO nanotips 12, and then biotinylated oligonucleotide willbe attached to the modified nanostructured surface as disclosed by G.Marrazza, I. Chianella, and M. Mascini, “Disposable DNA electrochemicalsensor for hybridization detection”, Biosens Bioelectron. 14, 43 (1999).The modified nanostructured surface includes the ZnO surface 12initially coated with Cr, Ti, etc., to help the subsequent Au layer wetthe surface. The Au film is then deposited and modified withthiol/dextran, which in turn will allow the covalent attachment ofavidin as disclosed by S. Tombelli, M. Mascini, L. Braccini, M.Anichini, and A. P. Turner, “Coupling of a DNA piezoelectric biosensorand polymerase chain reaction to detect apolipoprotein E polymorphisms”,Biosens Bioelectron. 15, 363 (2000). Thiolated DNA oligonucleotides arecovalently attached to mercaptosilane-derivatised surface viasuccinimidyl 4-[malemido-phenyl]butyrate (SMPB) crosslinker as disclosedby T. A. Taton, C. A. Mirkin, and R. L. Letsinge, “Scanometric DNA arraydetection with nanoparticle probes”, Science 289, 1757 (2000) and L. A.Chrisey, G. U. Lee, and C. E. O'Ferrall, “Covalent attachment ofsynthetic DNA to self-assembled monolayer films”, Nucleic Acids Res. 24,3031 (1996). The Thiolated DNA oligonucleotides can serve as thebiological recognition elements which recognize the analytes.

For testing the efficiency of DNA immobilization procedures, modeloligonucleotides with a radioisotope/fluorescent label are preferablyused. For testing and calibration of DNA/RNA ZnO nanotip biosensors, aseries of complementary pairs of oligonucleotides (20 and 50 nucleotidesin length) which are 30, 50 and 70% GC-reach are synthesized havingdifferent percents of complementarity (from no to several mismatches).Basically, preferably light with a particular wavelength (λ) is passedthrough the transparent ZnO nanotip 12 and one member of each pair ofoligonucleotides is immobilized on a surface of ZnO biosensor 10 and thedevice is tested in a series of hybridization experiments with thecorresponding targets. Different hybridization conditions, as well asdifferent RNA targets are evaluated. These targets may preferably belabeled. As an example of the practical application two sets ofexperiments are conducted with specific targets. One such target isdetection of cold-shock inducible cspA mRNA from E. coli. Some majoradvances in understanding of the regulatory mechanisms of cspAexpression have been made as disclosed by S. Phadtare, J. Alsina, and M.Inouye, “Cold-shock response and cold-shock proteins”, Curr OpinMicrobiol. 2, 175 (1999). Another is detection of mutations in the BRCA1gene. This gene is responsible for 40% of breast cancer cases and 80% ofbreast-ovarian cancer cases as disclosed by M. O. Nicoletto, M. Donach,A. D. Nicolo, G. Artioli, G. Banna, and S. Monfardini, “BRCA-1 andBRCA-2 mutations as prognostic factors in clinical practice and geneticcounseling”, Cancer Treat. Rev. 27, 295 (2001). We preferably useoligonucleotides complementary to the 185delAG and 188del11 mutations ofthe gene, which are the most common in all reported cases as disclosedby D. Tong, M. Stimpfl, A. Reinthaller, N. Vavra, S. Mullauer-Ertl, S.Leodolter, and R. Zeillinger, “BRCA1 gene mutations in sporadic ovariancarcinomas: detection by PCR and reverse allele specific oligonucleotidehybridization”, Clin. Chem. 45, 976 (1999). So, if we see binding, i.e.we get a signal such as fluorescent light, we know that patient's DNAstrength hits breast cancer mutation.

For protein immobilization, strategies similar to those of DNA are used.As mentioned above, proteins may have strong affinity to ZnO surface 12.The Au coated ZnO surface is modified with thiol/dextran and activatedby N-hydroxysuccinimide andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide allowing covalentattachment of the protein as disclosed by N. Barie and M. Rapp,“Covalent bound sensing layers on surface acoustic wave (SAW)biosensors”, Biosens Bioelectron 16, 979 (2001). The ZnO surface 12 willbe precoated with polyethylenimine and the protein will be crosslinkedto the surface via glutaraldehyde as disclosed by J. Ye, S. V. Letcher,and A. G. Rand, “Piezoelectric biosensor for the detection of Salmonellatyphimurium”, J. Food. Sci. 62, 1067 (1997). Alsobromocyano-immobilization will be preferably modified in which theshilding layer (polyimide or polystyrene) will be first loaded on theZnO surface 12, following CNBr activation and coupling the protein asdisclosed by T. Wessa, M. Rapp, and H. J. Ache, “New immobilizationmethod for SAW-biosensors: covalent attachment of antibodies via CNBr”,Biosens Bioelectron 14, 93 (1999).

In another case, for protein immobilization, a histidine kinase calledEnvZ was used. The protein was first phosphorylated with }-[³²P] ATP andsolution of ³²P-labled EnvZ was loaded on the nanotips grown on a squareglass plate (5×5 mm). After incubation for 90 minutes, plates werewashed extensively at room temperature by changing the washing buffersolution five times. The radioactivity of the plate was then measured.More than 90% of the protein was retained on the surface, indicating astrong affinity of the protein to the ZnO surface. It was also foundthat EnvZ attached on the nanotip surface retained its biochemicalproperty, as approximately 80% of ³²P radioactivity was released whenthe plate was incubated in a solution containing OmpR, an EnvZsubstrate, but no radioactivity was released in the presence of otherproteins. This model system is preferably used for analysis ofprotein-protein interactions via optical techniques, since EnvZ forms astoichiometric complex with OmpR.

Fluorescence resonance energy transfer (FRET) is a quantum mechanicalprocess wherein excitation energy is transferred from a donorfluorophore to an appropriately positioned acceptor positioned acceptorfluorophore without emission of a photon. Energy can be transferred thisway only over a very limited distance, and the efficiency of the energytransfer varies inversely with the sixth power of the distanceseparating the donor and acceptor. One of the most important uses ofFRET spectroscopy is to study protein-protein interactions.

For testing protein immobilization in addition of a histidine kinase weuse preferably as a model, calmodulin (CaM) fused with two mutantproteins, CFP (a mutant of green fluorescent protein, GFP; cyanfluorescent protein) at the N-terminal end and YFP (another mutant ofGFP; yellow fluorescent protein) at the C-terminal end as disclosed byA. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura,and R. Y. Tsien, “Fluorescent indicators for Ca2+ based on greenfluorescent proteins and calmodulin”, Nature 388, 882 (1997). Thisallows us to easily determine concentration of the protein bymeasurement of fluorescent intensity. Moreover, CaM protein is termed“cameleon”, since it responds to Ca⁺⁺ causing FRET (fluorescentresonance energy transfer) as mentioned above. It is critical to observewhether Ca⁺⁺ is close enough to calmodulin (CaM), in order for them tointeract. Again, preferably light with a particular λ (i.e. lightcapable of exciting the GFP) is passed through transparent ZnO nanotip12. If the green fluorescent protein fluoresces at its characteristic λ,there is no protein interaction since the protein distance is greaterthan a specific amount and therefore no FRET occurs. However, if thecalmodulin and Ca⁺⁺ are close enough (within FRET proximity) tointeract, the GFP is excited but does not emit a photon. Preferably, asensitized emission from Ca⁺⁺ occurs, so FRET occurs. The effect can bemonitored directly on the ZnO sensor 10. Since the ZnO film istransparent, the cameleon-bound ZnO film can be excited from the bottomand the intensity of the emission spectra recorded. The use of cameleonsallows us to evaluate the ZnO sensor 10 performance preferably by twomechanisms such as electrical (conductivity) and optical (fluorescence),or acoustic (SAW) and optical (fluorescence). The FRET method monitorsreal-time protein-protein interaction and/or conformational changes.Therefore, the comparison of our acoustic wave/conductivity measurementswith FRET data helps to evaluate signal responsiveness.

The ZnO DNA and protein nanotip array based sensors in this applicationwill further benefit to explore genome-wide gene expression formolecular diagnostics, drug target discovery, and validation of drugeffects. The ZnO nanotip-based biosensors can also be applied todevelopment of new methods for the prevention, diagnosis and treatmentof diseases. Furthermore, the application of ZnO and itsnanostructure-based biosensors can be extended to detection of toxicbiochemical agents and hazardous chemicals against bioterrorism andenvironmental monitoring and protection. Unlike other sensortechnologies, ZnO biosensors can operate in multimodes due to itsmultifunctional material properties (semiconductor, piezoelectric,transparent and conductive, etc.). Nanotips made from ZnO and itsternary compound can be used for UV absorption and fluorescencedetection. ZnO nanotip arrays can be highly dense for diagnostic kitsand flow-through systems, including ZnO UV biotesting bench (containingemitters, detectors, modulators, and filters), gene chip, lab-on-a chipand living-cell chip.

While the invention has been described in relation to the preferredembodiments with several examples, it will be understood by thoseskilled in the art that various changes may be made without deviatingfrom the fundamental nature and scope of the invention as defined in theappended claims.

1. A method for detecting biological molecules comprising: providing aconductivity mode ZnO nanotip biosensor including ZnO nanotips havingbinding sites including at least one biological probe; exposing saidbinding site to a sample having a potential target molecule; detecting achange in conductivity in said ZnO nanotips, wherein said change inconductivity being indicative of a chemical and biochemical reaction ofthe potential target molecule and the biological probe.
 2. The method ofclaim 1 wherein said ZnO nanotips serve for immobilization of DNA orprotein molecules to enhance detection of the corresponding targetedDNA, protein or small biomolecules.
 3. The method of claim 2 whereinsurface charge of the tips changes due to a biological and biochemicalreaction of the immobilized DNA, protein molecules or small biomoleculeson the ZnO nanotips with the corresponding targeted DNA or proteinmolecules.
 4. The method of claim 1 wherein said biosensor operates inmultiple modes due to multifunctional material properties of the ZnOnanotips such as semiconducting, piezoelectric, or transparent andcombinations thereof.
 5. A method for detecting biological moleculescomprising: providing a semiconductor FET biosensor including ZnOnanotips having binding sites including at least one biological probewherein said ZnO nanotips are deposited on gate region of the FET;exposing said binding site to a sample having a potential targetmolecule; detecting a change in conductivity in said ZnO nanotips,wherein said change in conductivity being indicative of a chemical andbiochemical reaction of the potential target molecule and the biologicalprobe.
 6. The method of claim 5 wherein said ZnO nanotips serve as DNAor protein molecule binding sites to detect presence of the DNA, proteinmolecules or small biomolecules to be targeted.
 7. The method of claim 6wherein upon said detection of the targeted molecules, a change occursin the surface charge of the ZnO nanotip-gate resulting in a change inthe channel conductance.
 8. The method of claim 5 wherein said FETbiosensor includes Si MOSFET.
 9. The method of claim 5 wherein said FETbiosensor includes GAs MESFET.
 10. A method for detecting biologicalmolecules comprising: providing a ZnO based SAW sensor including ZnOnanotips having binding sites including at least one biological probe;exposing said binding site to a sample having a potential targetmolecule; detecting a decrease in phase velocity under said ZnOnanotips, wherein said decrease in phase velocity being indicative of achemical and biochemical reaction of the potential target molecule andthe biological probe.
 11. The method of claim 10 wherein upon binding ofthe targeted molecules with said ZnO nanotips causes a mass loading on aSAW path resulting in said decrease of phase velocity under said ZnOnanotips.
 12. The method of claim 10 wherein said ZnO nanotips serve asDNA, protein molecule or small biomolecule binding sites.
 13. The methodof claim 12 wherein said ZnO nanotips enhance binding strength andimmobilization of the targeted DNA, protein molecules or smallbiomolecules.
 14. A method for detecting biological moleculescomprising: providing a ZnO based SAW sensor including ZnO nanotipshaving binding sites including at least one biological probe; exposingsaid binding site to a sample having a potential target molecule;detecting a change in UV absorption, wherein said change in UVabsorption being indicative of a chemical and biochemical reaction ofthe potential target molecule and the biological probe.
 15. The methodof claim 14 wherein said ZnO nanotips serve as DNA, protein molecule orsmall biomolecule binding sides.
 16. The method of claim 15 furtherincludes illuminating UV light on said sensor and the change in UVabsorption is detectable due to a biological and biochemical reaction ofsensor layer of immobilized DNA, protein molecules on the ZnO nanotipswith targeted DNA, protein molecules or small biomolecules.
 17. A methodfor detecting biological molecules comprising: providing a ZnO based BAWsensor including ZnO nanotips having binding sites including at leastone biological probe; exposing said binding site to a sample having apotential target molecule; detecting a change in resonance frequency ofthe sensor, wherein said change in resonance frequency being indicativeof a chemical and biochemical reaction of the potential target moleculeand the biological probe.
 18. The method of claim 17 wherein uponbinding of the targeted molecules with immobilized biomolecules on saidZnO nanotips causes a mass loading on the BAW resulting in the change inthe resonance frequency of the sensor.